Infection Of Blood Mononuclear Cells Research Articles

Detection of hepatitis C virus (HCV) proteins by immunofluorescence and HCV RNA genomic sequences by non-isotopic in situ hybridization in bone marrow and peripheral blood mononuclear cells of chronically HCV-infected patients.

Immunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36.4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. IF signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers.

An 85-aa segment of the GB virus type C NS5A phosphoprotein inhibits HIV-1 replication in CD4 + Jurkat T cells

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.

Clinical and Therapeutic Implications of Hepatitis C Virus Compartmentalization

Blood mononuclear cells (BMCs) frequently are infected by hepatitis C virus (HCV) variants that are not found in plasma. The influence of this compartmentalization on the natural and therapeutic outcome of hepatitis C is unknown. We studied 119 patients with previously untreated chronic HCV infection. Sixty-five of these patients started first-line treatment with pegylated interferon-alfa and ribavirin after enrollment in the study. The internal ribosomal entry site (IRES) of HCV RNA was amplified and compared between plasma and BMCs by means of single-strand conformational polymorphism (SSCP) analysis, line-probe assay, and cloning sequencing. The IRES SSCP patterns differed between plasma and BMCs in 54 (48%) of 113 assessable patients. Twenty-seven (24%) of these patients were co-infected by 2 HCV types or subtypes, only 1 of which was detectable in BMCs (n = 25) or in plasma (n = 2). SSCP-defined compartmentalization was more frequent in former drug users than in others (35/56 [60%] vs 19/56 [34%]; P < .01), and less frequent in patients with genotype 1 HCV in plasma (26/73 [24%] vs 28/40 [65%]; P < .01). The only variables that were independently predictive of a sustained virologic response were SSCP-defined compartmentalization (25/31 vs 10/32; P = .0001) and genotype 2 or 3 infection of BMCs (22/31 vs 8/34; P = .002). A significant proportion of patients with hepatitis C are co-infected by 2 or more HCV variants with distinct IRES sequences and distinct cellular tropism. This compartmentalization is a strong independent predictor of treatment efficacy.

Bluetongue Virus Infection of Bovine Monocytes

Cultures of adherent and non-adherent bovine blood mononuclear cells were infected with bluetongue virus (BTV) serotype 10. Production of BTV proteins in mononuclear cell cultures was detected by immune precipitation of viral proteins from [35S]methionine-labelled extracts of these cells, by immunofluorescence staining of cells using monoclonal antibodies (MAbs) to BTV proteins VP7 and NS2, and by flow cytometry with MAbs to VP2, VP7, NS1 and NS2. BTV-infected cells were most numerous in cultures of adherent mononuclear cells; infected cells were initially identified as monocytes on the basis of their morphology, and size and scatter characteristics as determined by analysis with a fluorescence-activated cell sorter (FACS). The majority of adherent mononuclear cells with these scatter characteristics were confirmed to be monocytes by FACS analysis with a MAb specific for bovine monocytes. Identification of BTV-infected adherent mononuclear cells as monocytes was further established by double immunofluorescent labelling, as infected adherent cells reacted with the MAb specific for bovine monocytes, and with another MAb specific for class II antigen. Infection of adherent mononuclear cells was also confirmed by transmission electron microscopy, as BTV virions and tubules were present in lysates of cultures of BTV-infected adherent mononuclear cells and within the cytoplasm of adherent cells. In contrast, BTV proteins were detected in few cells identified as lymphocytes on the basis of their scatter characteristics, and mean fluorescence of such cells was considerably less than that of BTV-infected monocytes. Viraemia persisted until 35 days after inoculation of a colostrum-deprived calf inoculated with BTV. Virus was isolated from blood mononuclear cells at 1 week after infection of the calf, but not thereafter. BTV infection of blood mononuclear cells was demonstrated until 9 days after inoculation by indirect immunofluorescence staining of mononuclear cells. In contrast, virus was consistently isolated from erythrocyte-enriched preparations throughout viraemia in titres comparable to those in whole blood. These results indicate that although bovine monocytes are readily infected in vitro with this strain of BTV serotype 10, infection of blood monocytes is unlikely to be responsible for the prolonged viraemia that consistently occurs in BTV-infected cattle.